Integration of a Reconstituted Cell-free Protein-synthesis System on a Glass Microchip
Integration of a Reconstituted Cell-free Protein-synthesis System on a Glass Microchip
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DOI：
10.2116/analsci.31.67
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发表时间：
2015-02
影响因子：
1.6
通讯作者：
Yo Tanaka;Y. Shimizu
中科院分区：
文献类型：
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作者：
Yo Tanaka;Y. Shimizu
最近，一种无细胞的蛋白质合成系统，仅从大肠杆菌翻译系统的基本要素重建，称为蛋白质合成使用重组元件（PURE），已被广泛用于合成生物学分析基本的生命系统。在这里，该系统被集成在玻璃微芯片系统上，以构建一个简单的蛋白质合成系统。通过链霉亲和素-生物素结合将GFP模板DNA固定在琼脂糖微珠上。将珠粒引入具有10 μm高坝结构的玻璃微芯片中的Y形微通道中，并使PURE系统反应混合物流过微通道。回收的溶液具有比引入微通道之前的反应混合物更高的荧光强度，从而证实已经实现GFP合成。固定有DNA微珠的微芯片可重复使用。这在简单和快速方面优于需要制备模板DNA或mRNA并将其添加到反应混合物中的常规体外蛋白质合成方案。
Recently, a cell-free protein synthesis system reconstituted solely from essential elements of the Escherichia coli translation system, termed protein synthesis using recombinant elements (PURE), has been widely used in synthetic biology to analyze fundamental life systems. Here, the system was integrated on a glass microchip system to construct a simple protein synthesis system. GFP template DNAs were immobilized on Sepharose microbeads by streptavidin-biotin binding. The beads were introduced into a Y-shaped microchannel in a glass microchip with a 10-μm height dam structure, and a PURE system reaction mixture was flowed through the microchannel. The recovered solutions had a higher fluorescent intensity than that of the reaction mixture before its introduction into the microchannel, thus verifying that GFP synthesis had been achieved. The microchip with DNA immobilized microbeads is reusable. This is advantageous over a conventional in vitro protein synthesis protocol requiring the preparation and addition of template DNA or mRNA into the reaction mixtures in aspect of simpleness and rapidness.